Ebola virus, a member of the family Filoviridae and the order Mononegavirales, is an enveloped, nonsegmented negative-strand RNA virus and is one of the most lethal human and nonhuman primate pathogens recognized to date (Feldmann et al., 1998; Vanderzanden et al., 1998). Four subtypes of Ebola virus have been identified, including Zaire, Sudan, Ivory Coast, and Reston (Sanchez et al., 1993). Human infection with subtype Zaire causes a fulminating, febrile, hemorrhagic disease that results in extensive mortality (Feldmann et al., 1993). Thus, Ebola virus infection presents a much-needed model to study virus-induced mechanisms leading to coagulation disorders and vascular instability. However, identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis.
Ebola virus particles have a filamentous appearance, but its shape may be branched, circular, U- or 6-shaped, or long and straight (Feldmann et al., 1996). Virions show a uniform diameter of approximately 80 nm, but vary greatly in length. Ebola virus particles consist of seven structural proteins. The glycoprotein (GP) of Ebola virus forms spikes of approximately 7 nm, which are spaced at 5- to 10-nm intervals on the virion surface (Feldmann et al., 1996 and Peters et al., 1995). Cleavage of the GP is thought to be an important determinant of viral pathogenicity (Volchkov et al., 1998; Sanchez et al., 1996; Takada et al., 1997; Volchkov et al., 1998a; Volchkov et al., 1998b; Wool-Lewis et al., 1998; Yang et al., 2000). The Ebola virus GP contains a highly conserved consensus motif for the subtilisin-like endoprotease furin, and previous studies demonstrated GP cleavage by this protease (Nina et al., 1991). Nonetheless, studies of murine leukemia virus (Wood-Lewis et al., 1999) or vesicular stomatitis virus (VSV) (Ito et al., 2001) pseudotyped with mutant Ebola virus GPs lacking the furin recognition motif at the cleavage site, showed that GP cleavage by furin was not essential for infectivity of the pseudotyped viruses. In many viruses, GP cleavage by furin and related endoproteases is essential for their infectivity. Thus, the significance of GP cleavage for the Ebola virus life cycle remains in question.
GP is the only transmembrane protein of Ebola virus, and is responsible for receptor binding and membrane fusion (Takada et al., 1997). Cells infected with recombinant vaccinia virus expressing the GP produced virosomes that varied in shape and diameter but uniformly possessed spike structures on their surface (Volchkov et al., 1998c), although the effects of over 80 vaccinia viral proteins (Moss, 1995) on the formation of particles are unknown. Similar virosomes are also released from Ebola virus-infected cells (Volchkov et al., 1998c). These findings suggest that the GP contributes not only to an early stage of the viral infection cycle but also to viral budding.
In addition, although recent studies have begun to address the immune response to viral infection (Baize et al., 1999; Basler et al., 2000; Vanderzanden et al., 1998; and Wilson et al., 2000), as well as the functions of the viral proteins involved in the replicative process (VP30, VP35, NP, L) (Basler et al., 2000 and Muhlberger et al., 1999) and GP, little is known about the functions of the viral proteins associated with the membrane, including viral protein 40 (VP40), which appears equivalent to matrix protein of other viruses.
The matrix proteins of many nonsegmented, negative-sense RNA viruses play a critical role in viral particle formation (virus assembly) and budding (Garoff et al., 1998). Expression of the matrix protein of VSV in insect and mammalian cells results in evagination of matrix protein-containing vesicles from the plasma membrane surface (Justice et al., 1995; and Li et al., 1993). Matrix proteins interact with membranes in a hydrophobic and/or electrostatic manner and electron micrographs of nonsegmented, negative-sense RNA viruses have demonstrated that the matrix protein forms a layer associated with the inner leaflet of the lipid bilayer (Garoff et al., 1998).
VP40 is the most abundant protein in virions (it represents 38% of the protein in the viral particle) and is located beneath the viral membrane, where is presumably maintains the structural integrity of the particle (Feldmann et al., 1996). VP40 is encoded by the third gene in the linear 3′-5′ RNA genome of Ebola virus and is 326 amino acids in length, which includes a number of hydrophobic regions (Elliott et al., 1985 and Sanchez et al., 1996). VP40 contains a PPXY motif (SEQ ID NO:50; X denotes any amino acid) at amino acids 10-13 (Harty et al., 1996) that is also present at amino acids 16-19 in Marburg virus, strain Popp (Sanchez et al., 1993). This motif has been shown to play an important role in the budding of rabies virus and VSV: when either of the prolines or the tyrosine of this motif is altered in the matrix proteins of these viruses, viral budding is markedly reduced by comparison to findings with wild-type virus (Harty et al., 1996). Mutation of the PPXY (SEQ ID NO:50) motif in the matrix protein of VSV appears to reduce virus yield by pre-empting budding of assembled virions at the plasma membrane (Jayakar et al., 2000). This motif interacts with the WW domain found in many cellular regulatory and signal transduction proteins (Bork et al., 1994 and Chen et al., 1995) and interactions between one or more cellular proteins and the matrix proteins of these viruses are thought to be crucial for efficient virus release from cells (Harty et al., 1999).
The matrix proteins of many enveloped viruses are thought to interact with the cytoplasmic tails of viral glycoproteins. Such interaction is believed to be important for virus assembly. In influenza viruses, the removal of the cytoplasmic tail of the hemagglutinin or neuraminidase glycoprotein alters virion morphology (Jin et al., 1997; Mitnaul et al., 1996). Although not essential for normal particle formation in rabies virus and VSV, glycoproteins enhance the efficiency of particle formation (Mebatsion et al., 1996; Mebatsion et al., 1999; Schnell et al., 1998).
Thus, what is needed is a method to readily manipulate the filovirus genome.